Live imaging analysis of human gastric epithelial spheroids reveals spontaneous rupture, rotation and fusion events

Three-dimensional cultures of primary epithelial cells including organoids, enteroids and epithelial spheroids have become increasingly popular for studies of gastrointestinal development, mucosal immunology and epithelial infection. However, little is known about the behavior of these complex cultures in their three-dimensional culture matrix. Therefore, we performed extended time-lapse imaging analysis (up to 4 days) of human gastric epithelial spheroids generated from adult tissue samples in order to visualize the dynamics of the spheroids in detail. Human gastric epithelial spheroids cultured in our laboratory grew to an average diameter of 443.9 ± 34.6 μm after 12 days, with the largest spheroids reaching diameters of >1000 μm. Live imaging analysis revealed that spheroid growth was associated with cyclic rupture of the epithelial shell at a frequency of 0.32 ± 0.1/day, which led to the release of luminal contents. Spheroid rupture usually resulted in an initial collapse, followed by spontaneous re-formation of the spheres. Moreover, spheroids frequently rotated around their axes within the Matrigel matrix, possibly propelled by basolateral pseudopodia-like formations of the epithelial cells. Interestingly, adjacent spheroids occasionally underwent luminal fusion, as visualized by injection of individual spheroids with FITC-Dextran (4 kDa). In summary, our analysis revealed unexpected dynamics in human gastric spheroids that challenge our current view of cultured epithelia as static entities and that may need to be considered when performing spheroid infection experiments.